H.O. Halvorson

Intracellular protein and nucleic acid turnover in resting yeast cells

Abstract In the absence of exogenous energy and nitrogen, resting yeast cells are capable of replenishing both their nucleotide and free amino acid pools. This replenishment phenomenon is the result of intracellular protein and nucleic acid breakdown rather than of cell lysis. Isotopically labeled cells showed turnover rates of 6.6 · 10−3 h−1 and 1.5 · 10−3 h−1 for protein and nucleic acid respectively. Protein and nucleic acid degradation, as well as their synthesis, are energy-requiring reactions. When exogenous energy is available, the degradation products are reutilized for protein and nucleic acid synthesis. Employing 15N-purine-grown cells, it was found that the amino groups of nucleic acid purines can serve as nitrogen reservoirs for limited protein synthesis.

Separation of labeled from unlabeled proteins by equilibrium density gradient sedimentation

Abstract If equilibrium density gradient ultracentrifuge experiments are conducted under carefully designed conditions, the resolution of the method can be markedly improved or, alternatively, the experiment can be designed to describe the nature and amount of many widely varying macromolecules in a single experiment. The theoretical limits on resolution are examined for two types of experiments. Solutes have been found which give high resolution and appropriate densities for protein, RNA, and DNA equilibrium centrifugation. The properties of these solutes are tabulated. The theory has been tested by separation of normal β-galactosidase from samples labeled with N 15 , C 13 , and deuterium. It is shown that the method will resolve labeled from unlabeled proteins if an adequate, but attainable, density difference has been introduced.

Studies on protein and nucleic acid turnover in growing cultures of yeast.

Abstract A study of the utilization of individually 14C-labeled aminoc acid and nucleotide pools in exponentially growing yeast indicated that precursors are converted essentially irreversibly into proteins and nucleic acids. The maximal breakdown rates for each were 0.01 h−1. Employing a more sensitive method, involving an internal nucleic acid purine trap for protein degradation products, the protein breakdown rate was estimated at 2.8· 10−4 h−1. The proteins therefore have an average half-life of 18 days or longer in cells dividing every 90 minutes. The data indicate that protein breakdown in growing cells is only 4% of that in resting cells. The significance of the difference in protein and possibly nucleic acid stabilities of growing and resting cells is discussed.

Turnover of protein and nucleic acid in soluble and ribosome fractions of non-growing Escherichia coli

Abstract The stability of protein and of RNA has been examined in the ribosome and soluble fractions of non-growing E. coli . In both fractions there is balanced degradation and re-synthesis of protein at about 5% per h. The RNA of the ribosomes is also degraded at this rate but re-synthesized at only 1.5% per h. In the soluble fraction the rate of nucleic acid synthesis exceeds the rate of degradation, so that there appears to be net transfer of material from the ribosomes to the soluble fraction. Degradation of ribosomes supplies much of the free amino acids and almost all of the ribonucleotides passing through the free pool during starvation.

Uptake of α-thioethyl D-glucopyranoside by Saccharomyces cerevisiae I. The genetic control of facilitated diffusion and active transport

Abstract α-Thioethyl D -glucopyranoside has been shown to enter Saccharomyces cerevisiae by two mechanisms, facilitated diffusion and active transport, both controlled by the MG 2 gene. Only facilitated diffusion was observed in glucose-grown strains whereas in cells induced by α-thioethyl D -glucopyranoside or α-methyl glucoside, α-thioethyl D -glucopyranoside was accumulated against a concentration gradient. The induction of the active transport system was completely inhibited by 0.1 M acetate. The K m value of α-thioethyl D -glucopyranoside for facilitated diffusion was calculated as 50 mM.

Uptake of α-thioethyl D-glucopyranoside by Saccharomyces cerevisiae II. General characteristics of an active transport system

Abstract The properties of α-thioethyl d -glucopyranoside accumulation in induced cells of Sacchromyces cerevisiae carrying the dominant MG2 gene have been studied. At equilibrium the internal concentration of α-thioethyl- d -glucopyranoside was 10–150 times that of the external media depending upon the external concentration of α-thioethyl- d -glucopyranoside employed. The uptake system was stereospecific, had a pH optimum of 4.6 and displayed a Michaelis-Menten substrate dependence with a Km of 1.8 mM. Accumulated α-thioethyl- d -glucopyranoside could be displaced by maltose, glucose or α-methyl glucoside but not by NaN3, an effective inhibitor of α-thioethyl- d -glucopyranoside uptake. The relationships of the α-thioethyl- d -glucopyranoside pyranoside uptake systems by facilitated diffusion in uninduced cells and by active transport in induced cells are discussed.

Comparison of the α-glucosidases of saccharomyces produced in response to five non-allelic maltose genes

Abstract A comparison has been undertaken of the α-glucosidases produced in Saccharomyces in response to 5 non-allelic maltose genes. The partially purified enzymes were found to be indistinguishable in regard to heat inactivation, electrophoretic mobility, chromatography from CM-cellulose or DEAE-cellulose columns, neutralization with specific antiserum, or substrate specificity. In each of the genotypes only a single species of α-glucosidase was observed.

The specificity of induction of β-glucosidase in Saccharomyces cerevisiae

Abstract The kinetics and stereospecificity of gratuitos induction of β-glucosidase in S. cerevisiae strain Yeast Foam have been studied. Enzyme synthesis, following the addition of either methyl- or ethyl-β- d -thioglucoside as inducers, is constant and proportional to increases in cell mass. The differential rate of enzyme synthesis is dependent upon the concn. and type of the inducer. A comparison of the stereospecificity of the induction process and of the enzyme itself indicates that induction is independent of enzyme action. Phenyl-β- d -thioglucoside was found to be a competitive inhibitor of enzyme induction.

Intermediate metabolism of aerobic spores. V. The purification and properties of l-alanine dehydrogenase

Abstract An l -alanine dehydrogenase (AID) has been purified from extracts of spores of Bacillus cereus strain T. This enzyme catalyzes the reversible reaction: l -alanine + DPN+ + H2O ⇌ pyruvate + NH3 + DPNH + H+ The enzyme is specific for DPN and is inhibited by heavy metals and sulfhydrylbinding agents. The pH optimum for deamination is 9.8 and for amination 8.8. The equilibrium constant is 1.36 × 10−14. The properties of the enzyme are identical to those previously described for the AID of vegetative cells of Bacillus. In spores, AID is the primary route of l -alanine deamination.

The nucleases of yeast. I. Properties and variability of ribonucleases.

Abstract 1. 1.Nucleases are located in both ribosomes and supernatant of the yeast, Saccharomyces fragilis X Saccharomyces dodzhanskii. Ribosomes prepared from either exponentially growing cells or stationary cells exhibit nuclease activity with a pH optimum of around 7 which is not inhibited by 0.02 M EDTA, Polyvinylsulfate inhibits only at high concentrations. Nuclease activity in the 105 000 × g supernatant from exponentially growing cells has a pH optimum of around 8 and is severely inhibited by EDTA and polyvinylsulfate. In contrast, supernant nuclease from stationary cells has a pH optimum of around 6 and is only partially inhibited by EDTA. 2. 2.Supernant nuclear I and II from logarithmic cells have been partially purified. Their pH optimums are 7.4 and 7.6, respectively. Both nucleases are inhibited by EDTA, Zn2+, polyvinylsulfate and phosphate. The inhibition of EDTA is reversed by the addition of Mg2+. Polyribonucleotides are hydrolysed to di- and trioligonucleotides with 5′-phosphomonoester end groups by both nucleases.